Humanized anti-cd22 recombinant immunotoxin and application thereof

ABSTRACT

Provided is a humanized anti-CD22 recombinant immunotoxin, which has reduced immunogenicity and maintains the CD22 binding affinity and corresponding biological activity before humanization transformation, and is used for preparing a drug for treating CD22-related B cell malignant tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase Patent Application ofInternational Application Number PCT/CN2021/077461, filed on Feb. 23,2021, which claims the priority benefit of Chinese Patent ApplicationNo. CN202011048723.0 filed on 29 Sep. 2020, each of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted in ASCIIformat via EFS-Web and is incorporated herein by reference in itsentirety. Said ASCII copy, which was last modified on Mar. 23, 2023, isnamed “LC21210002P20230315SequnceListing.txt”, and is 53,727 bytes insize.

TECHNICAL FIELD

The present invention relates to the field of biomedicines, inparticular to a recombinant immunotoxin of humanized anti-CD22 antibodyfragment fused with truncated pseudomonas exotoxin A and applicationthereof.

BACKGROUND OF THE INVENTION

CD22 (Siglec-2) is a sialic acid-binding immunoglobulin-like lectin(Siglec) receptor, that binds specifically to sialic acid(Sia)-containing glycans, facilitating cell adhesion and/or cellsignaling^([1]). CD22 expression is restricted to B cells and plays acritical role in establishing a baseline level of B-cell inhibition, andthus is a critical determinant of homeostasis in humoral immunity. CD22is expressed in neoplastic cells in various proportions of B-cellmalignancies, including B-lymphoblastic leukemia/lymphomas and matureB-cell leukemia/lymphomas^([2]). The expression of CD22 can beparticularly strong in hairy cell leukemia (HCL) and prolymphocyticleukemia.

Hairy cell leukemia (HCL), first described by Bouroncle and colleaguesin 1958, is a chronic malignancy of mature neoplastic B cells with acharacteristic serrated cytoplasmic border^([3,4]). HCL accounts for 2%of all leukemias in the USA (500-800 new cases in the United States peryear^([5])) and is characterized by pancytopenia, and splenomegaly.Purine analogues (cladribine or pentostatin) are the standard of carefor initial treatment for HCL and are associated with durable remissionsthat last for years; however, many patients relapse and requireadditional therapy ^([6]). Subsequent treatment is usually with arevised regimen of purine analogues, although treatment efficacy isreduced, patients have shorter remissions and are ultimately refractoryto treatment^([7]). Moreover, purine analogues have been associated withneurotoxicity ^([8]) and are very immunosuppressive, which may increasethe risk of opportunistic infections^([4]).

Immunotoxins have been proven to be effective therapeutics for hairycell leukemia. Immunotoxins are antibody-conjugated therapeutics thattarget and kill cancer cells using a potent cytotoxic payload, such asbacterial toxins ^([9]), plant-derived toxins ^([10]), and syntheticchemicals ^([11]). The first generation of the anti-CD22 immunotoxin wasdeveloped at the National Cancer Institute in the late 1990s, reportedby the name of BL22 (RFB4(dsFv)-PE38, or CAT-3888). It employed a murineanti-CD22 antibody fused to a Pseudomonas exotoxin A (PE38), which is a38 kDa fragment of Pseudomonas exotoxin A. CAT-3888 entered a phase Itrial and reported remissions for leukemia in 2001 ^([12-16]). However,CAT-3888 was succeeded by moxetumomab pasudotox (HA22, or CAT-8015), anupdated immunotoxin comprising modifications in the PE38 and theanti-CD22 antibody fragment. HA22 made changes to three amino acids inthe antibody fragment, comparing to CAT-3888, to increase the bindingaffinity for the target molecule.

HA22 is composed of the Fv fragment of a murine anti-CD22 monoclonalantibody fused to PE38. Mechanistically, the Fv portion of HA22 binds toCD22, which is a cell surface receptor expressed on a variety ofmalignant B cells, thereby delivering the toxin moiety PE38 directly totumor cells. Once internalized, PE38 catalyzes the ADP ribosylation ofthe diphthamide residue in elongation factor-2 (EF-2), resulting in therapid fall in levels of the anti-apoptotic protein myeloid cell leukemia1 (McI-1), leading to apoptotic cell death ^([17, 18]). HA22 wasapproved by the U.S. FDA in 2018 for the treatment of adults withrelapsed or refractory hairy cell leukemia who received at least twoprior systemic therapies, including treatment with a purine nucleosideanalogue.

The early immunotoxin designs lacked a sufficient therapeutic window towarrant further clinical development, and among which, the inherentimmunogenicity and rapidly development of antidrug antibodies was a keyfactor ^([19]). The targeting portion contained in HA22, i.e., the Fvportion of the murine anti-CD22 monoclonal antibody has certainimmunogenicity in human since it is murine derived, and thereby causesthe generation of undesirable neutralizing anti-drug antibodies (ADAs),and adversely influences pharmacokinetics profiles, includinginfluencing activity of the drug at given dosages, acceleratingclearance of the drug in vivo, and limiting times and effectiveness ofrepeated dosing.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, an object of the presentinvention is to provide a novel recombinant immunotoxin based onpseudomonas exotoxin A. In the recombinant immunotoxin, the pseudomonasexotoxin A is fused with a humanized anti-CD22 monoclonal antibodyfragment which will have reduced immunogenicity and retain the affinityfor the target CD22, compared with the original murine-derived anti-CD22monoclonal antibody fragment; at the same time, the novel recombinantimmunotoxin formed will have a strong immunotoxin efficacy, e.g., growthinhibitory and apoptotic effects on CD22 expressing tumor cells.

A further object of the present invention is to provide the use of thenovel recombinant immunotoxin.

Technical solutions of the present invention are as follows.

In one aspect, the present disclosure provides a humanized anti-CD22recombinant immunotoxin which is a polypeptide molecule comprising twopolypeptide chains as follows: (1) a first polypeptide chain, comprisinga light chain variable region (V_(L)) of an anti-CD22 antibody, whereinthe light chain variable region (V_(L)) comprises an amino acid sequenceas shown in SEQ ID NO. 16 or SEQ ID NO. 18 or an amino acid sequencehomologue thereof having at least 75% sequence identity to the aminoacid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18;

(2) a second polypeptide chain, comprising a heavy chain variable region(V_(H)) of an anti-CD22 antibody and a cytotoxin directly or indirectlylinked to the heavy chain variable region (V_(H)), wherein the heavychain variable region (V_(H)) comprises an amino acid sequence as shownin SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acid sequence homologuethereof having at least 75% identity to the amino acid sequence as shownin SEQ ID NO. 3 or SEQ ID NO. 4.

As used herein, the “amino acid sequence homologue” refers to an aminoacid sequence derived from a corresponding amino acid sequence buthaving one or more amino acid substitutions, additions, or deletionsrelative thereto.

In the first polypeptide chain, preferably, the amino acid sequencehomologue has at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18. Furtherpreferably, the amino acid sequence homologue comprises the amino acidsequences as shown in SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33,and has the same amino acid residues as those in SEQ ID NO. 16 or SEQ IDNO. 18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70 and 72corresponding to the amino acid sequence as shown in SEQ ID NO. 14 (theamino acid positions are numbered according to the amino acid sequenceas shown in SEQ ID NO. 14), but is not the amino acid sequence as shownin SEQ ID NO. 14.

In the second polypeptide chain, preferably, the amino acid sequencehomologue has at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4. Furtherpreferably, the amino acid sequence homologue comprises the amino acidsequences as shown in SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30,and has the same amino acid residues as those in SEQ ID NO. 3 or SEQ IDNO. 4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80 correspondingto the amino acid sequence as shown in SEQ ID NO. 2 (the amino acidpositions are numbered according to the amino acid sequence as shown inSEQ ID NO. 2), but is not the amino acid sequence as shown in SEQ ID NO.2.

Preferably, in the humanized anti-CD22 recombinant immunotoxin accordingto the present invention, the light chain variable region (V_(L)) in thefirst polypeptide chain comprises the amino acid sequence as shown inSEQ ID NO. 16 or the amino acid sequence homologue thereof; and theheavy chain variable region (V_(H)) in the second polypeptide chaincomprises the amino acid sequence as shown in SEQ ID NO. 3 or the aminoacid sequence homologue thereof.

Preferably, in the humanized anti-CD22 recombinant immunotoxin accordingto the present invention, the light chain variable region (V_(L)) in thefirst polypeptide chain comprises the amino acid sequence as shown inSEQ ID NO. 18 or the amino acid sequence homologue thereof; and theheavy chain variable region (V_(H)) in the second polypeptide chaincomprises the amino acid sequence as shown in SEQ ID NO. 3 or the aminoacid sequence homologue thereof.

Preferably, in the humanized anti-CD22 recombinant immunotoxin accordingto the present invention, the light chain variable region (V_(L)) in thefirst polypeptide chain comprises the amino acid sequence as shown inSEQ ID NO. 16 or the amino acid sequence homologue thereof; and theheavy chain variable region (V_(H)) in the second polypeptide chaincomprises the amino acid sequence as shown in SEQ ID NO. 4 or the aminoacid sequence homologue thereof.

Preferably, in the humanized anti-CD22 recombinant immunotoxin accordingto the present invention, the light chain variable region (V_(L)) in thefirst polypeptide chain and the heavy chain variable region (V_(H)) inthe second polypeptide chain are covalently linked, e.g., via adisulfide bond.

Preferably, in the humanized anti-CD22 recombinant immunotoxin accordingto the present invention, the cytotoxin in the second polypeptide chainis pseudomonas exotoxin A or a mutant or fragment of the pseudomonasexotoxin A with cytotoxicity retained.

Natural occurring pseudomonas exotoxin A (PE) is a bacterial toxinsecreted by Pseudomonas aeruginosa. It is a monomeric protein with amolecular weight of 66 kD. PE molecule can be modified, or part of itssequence can be removed, to reduce or eliminate nonspecific binding ofthe toxin while maintaining cytotoxicity. For example, a mutant ofpseudomonas exotoxin A is a modified pseudomonas exotoxin A that retainscytotoxicity, and the modification may be conservative modification. Forexample, a mutant has at least 75%, preferably at least 80%, furtherpreferably at least 90%, 91%, 92%, 93%, 94%, 95% amino acid sequenceidentity to unmodified natural PE. Also, for example, a fragment ofpseudomonas exotoxin A is a truncated form of pseudomonas exotoxin Athat retains cytotoxicity. Preferably, the mutant or fragment ofpseudomonas exotoxin A according to the present invention is, such as,PE40, PE38, PE35, PE24, mPE24, or T19, T20, M-11^([21, 22, 23, 24, 25]).According to one particular embodiment of the present invention, thefragment is PE38 with amino acid sequence as shown in SEQ ID NO. 1.

Preferably, in the second polypeptide chain, the cytotoxin is fused tothe C-terminus of the heavy chain variable region (V_(H)) of theanti-CD22 antibody. For example, the N-terminus of the cytotoxin isfused, directly or via a linker, to the C-terminus of the heavy chainvariable region (V_(H)). The linker can form covalent bonds with theheavy chain variable region (V_(H)) and with the cytotoxin respectively,without affecting the function of each functional component in thehumanized anti-CD22 recombinant immunotoxin. Linkers suitable are knownin the art, such as linear or branched carbon linkers, heterocycliccarbon linkers, and peptide linkers. In the humanized anti-CD22recombinant immunotoxin according to the present invention, preferably,no linker is used, or a peptide linker i.e. a linking peptide is used,such as a flexible linking peptide comprising one or more (GGGGS) aminoacid sequences.

In the humanized anti-CD22 recombinant immunotoxin according to thepresent invention, the first polypeptide chain and the secondpolypeptide chain can constitute a form of Fab, that is, the firstpolypeptide chain further comprises a kappa light chain constant region,preferably a human kappa light chain constant region fused to theC-terminus of the light chain variable region (V_(L)); alternatively,the second polypeptide chain further comprises a heavy chain constantregion CH1, preferably a human heavy chain constant region CH1 fused tothe C-terminus of the heavy chain variable region (V_(H)).

According to one particular embodiment of the present invention, in thehumanized anti-CD22 recombinant immunotoxin according to the presentinvention, the first polypeptide chain comprises the amino acid sequenceas shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof,and the second polypeptide chain comprises the amino acid sequence asshown in SEQ ID NO. 9 or the amino acid sequence homolog thereof.

Alternatively, the first polypeptide chain comprises the amino acidsequence as shown in SEQ ID NO. 18 or the amino acid sequence homologuethereof, and the second polypeptide chain comprises the amino acidsequence as shown in SEQ ID NO. 9 or the amino acid sequence homologuethereof.

Alternatively, the first polypeptide chain comprises the amino acidsequence as shown in SEQ ID NO. 16 or the amino acid sequence homologuethereof, and the second polypeptide chain comprises the amino acidsequence as shown in SEQ ID NO. 10 or the amino acid sequence homologuethereof.

In another aspect, the present disclosure provides a nucleic acidmolecule comprising a nucleotide sequence encoding the first polypeptidechain in the humanized anti-CD22 recombinant immunotoxin according tothe present invention and/or a nucleotide sequence encoding the secondpolypeptide chain in the humanized anti-CD22 recombinant immunotoxinaccording to the present invention. The nucleic acid molecule accordingto the present invention may be a single nucleotide sequence encodingthe first polypeptide chain or the second polypeptide chain; or, thenucleic acid molecule according to the present invention may be a singlenucleotide sequence encoding both the first polypeptide chain and thesecond polypeptide chain. Alternatively, the nucleic acid moleculeaccording to the present invention may be a combination of twonucleotide sequences, encoding the first polypeptide chain and thesecond polypeptide chain, respectively.

In yet another aspect, the present disclosure provides a vectorcomprising the nucleic acid molecule according to the present invention.The vector can be a eukaryotic expression vector, a prokaryoticexpression vector, an artificial chromosome, a phage vector, or thelike. According to one particular embodiment of the present invention,the vector is a prokaryotic expression vector, such as an expressionplasmid comprising the nucleic acid molecule.

Maps of plasmids according to one particular embodiment of the presentinvention containing the nucleotide sequences encoding the firstpolypeptide chain and the second polypeptide chain are shown in FIG. 3 .

In yet another aspect, the present disclosure provides a host cell. Thehost cell is transformed or transfected with the nucleic acid moleculeand/or the vector according to the present invention, and thus containsthe nucleic acid molecule and/or vector of the present invention, forpreservation or expression. The host cell can be any prokaryotic oreukaryotic cell, such as a bacterial or insect, fungal, plant or animalcell. According to one particular embodiment of the present invention,the host cell is a prokaryotic cell, such as E. coli cells.

According to the disclosure of the present invention, the humanizedanti-CD22 recombinant immunotoxin, the nucleic acid molecule, the vectorand/or the host cell according to the present invention can be obtainedby utilizing any conventional techniques known in the art. For example,the first polypeptide chain and the second polypeptide chain can beexpressed in a host cell with the nucleic acid molecule or vectoraccording to the present invention, and then a heterodimer, in which thelight chain variable region (V_(L)) in the first polypeptide chain islinked to the heavy chain variable region (V_(H)) in the secondpolypeptide chain via a disulfide bond, can be formed through recoveryand in vitro refolding of the chains.

The humanized anti-CD22 recombinant immunotoxin, the nucleic acidmolecule, the vector and/or the host cell according to the presentinvention can be contained in a composition, for example, apharmaceutical composition, more particularly in a pharmaceuticalpreparation, to be used for various purposes as actually needed.

Thus, in still a further aspect, the present disclosure also provides acomposition comprising the humanized anti-CD22 recombinant immunotoxin,the nucleic acid molecule, the vector and/or the host cell according tothe present invention. Preferably, the composition is a pharmaceuticalcomposition which optionally comprises a pharmaceutically acceptableexcipient.

The present disclosure further provides following related uses of thesubject matters described above based on the humanized anti-CD22recombinant immunotoxin which is capable of binding to target CD22,especially human CD22, a B cell surface marker.

Specifically, in one aspect, the present disclosure provides use of thehumanized anti-CD22 recombinant immunotoxin, the nucleic acid molecule,the vector, the host cell or the composition in the manufacture of amedicament for the treatment of a CD22-related B cell malignant tumor.

Preferably, the CD22-related B-cell malignant tumor is a B-cellmalignant tumor characterized by high CD22 expression, such as alymphoma or leukemia with high CD22 expression. Preferably, the lymphomais non-Hodgkin's lymphoma, small lymphocytic lymphoma or mantle celllymphoma; and the leukemia is chronic lymphocytic leukemia, hairy cellleukemia or acute lymphocytic leukemia.

In another aspect, the present disclosure provides a method for treatinga CD22-related B-cell malignant tumor, comprising administering to asubject in need thereof the humanized anti-CD22 recombinant immunotoxin,the nucleic acid molecule, the vector and/or the host cell. The subjectis a mammal, including human or non-human primate, and a domestic,livestock or laboratory mammal such as a dog, a cat, a cow, a pig, asheep, a horse, a murine, and a rabbit. Preferably, the subject is ahuman.

The humanized V_(H) and V_(L) according to the present invention canalso form a separate dsFv antibody, so in yet another aspect, thepresent disclosure provides a humanized anti-CD22 antibody or antibodyfragment, the antibody or antibody fragment comprising a heavy chainvariable region (V_(H)) and a light chain variable region (V_(L)),wherein the heavy chain variable region (V_(H)) comprises an amino acidsequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acidsequence homologue thereof having at least 75% identity to the aminoacid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4, and the lightchain variable region (V_(L)) comprises an amino acid sequence as shownin SEQ ID NO. 16 or SEQ ID NO. 18 or an amino acid sequence homologuethereof having at least 75% identity to the amino acid sequence as shownin SEQ ID NO. 16 or SEQ ID NO. 18.

In the light chain variable region (V_(L)), preferably, the amino acidsequence homologue has at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99/o sequenceidentity to the amino acid sequence as shown in SEQ ID NO. 16 or SEQ IDNO. 18. Further preferably, the amino acid sequence homologue comprisesthe amino acid sequences as shown in SEQ ID NO. 31, SEQ ID NO. 32 andSEQ ID NO. 33, and has the same amino acid residues as those in SEQ IDNO. 16 or SEQ ID NO. 18 at positions 3, 5, 36, 37, 47, 48, 49, 50, 65,67, 69, 70 and 72 corresponding to the amino acid sequence as shown inSEQ ID NO. 14 (the amino acid positions are numbered according to theamino acid sequence as shown in SEQ ID NO. 14), but is not the aminoacid sequence as shown in SEQ ID NO. 14.

In the heavy chain variable region (V_(H)), preferably, the amino acidsequence homologue has at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4.Further preferably, the amino acid sequence homologue comprises theamino acid sequences as shown in SEQ ID NO. 28, SEQ ID NO. 29 and SEQ IDNO. 30, and has the same amino acid residues as those in SEQ ID NO. 3 orSEQ ID NO. 4 at positions 3, 48, 49, 50, 69, 71, 73, 75, and 80corresponding to the amino acid sequence as shown in SEQ ID NO. 2 (theamino acid positions are numbered according to the amino acid sequenceas shown in SEQ ID NO. 2), but is not the amino acid sequence as shownin SEQ ID NO. 2.

Preferably, in the humanized anti-CD22 antibody or antibody fragmentaccording to the present invention, the light chain variable region(V_(L)) comprises the amino acid sequence as shown in SEQ ID NO. 16 orthe amino acid sequence homologue thereof, and the heavy chain variableregion (V_(H)) comprises the amino acid sequence as shown in SEQ ID NO.3 or the amino acid sequence homologue thereof.

Preferably, in the humanized anti-CD22 antibody or antibody fragmentaccording to the present invention, the light chain variable region(V_(L)) comprises the amino acid sequence as shown in SEQ ID NO. 18 orthe amino acid sequence homologue thereof; and the heavy chain variableregion (V_(H)) comprises the amino acid sequence as shown in SEQ ID NO.3 or the amino acid sequence homologue thereof.

Preferably, in the humanized anti-CD22 antibody or antibody fragmentaccording to the present invention, the light chain variable region(V_(L)) comprises the amino acid sequence as shown in SEQ ID NO. 16 orthe amino acid sequence homologue thereof; and the heavy chain variableregion (V_(H)) comprises the amino acid sequence as shown in SEQ ID NO.4 or the amino acid sequence homologue thereof.

Preferably, the humanized anti-CD22 antibody according to the presentinvention is a monoclonal antibody, which further comprises a humanheavy chain constant region (CH) and a light chain constant region (CL);preferably, the monoclonal antibody comprises a heavy chain (HC) and alight chain (LC).

Depending on the heavy chain constant region sequence of the monoclonalantibody, the antibody can be of IgA, IgD, IgE, IgG or IgM class, evenfurther of a subclass (isotype) such as IgG1, IgG2, IgG3, or IgG4.Depending on the light chain constant region sequence of the monoclonalantibody, the antibody can be of kappa type or lambda type.

Preferably, the antibody fragment according to the present invention isa monoclonal antibody fragment, preferably Fab, Fab′, F(ab′)2, scFv,dsFv or any other fragment of the antibody that retains the ability torecognize or bind to CD22. Depending on the context, “antibody fragment”in the present disclosure can be used interchangeably with “antibody”.

More preferably, the antibody fragment according to the presentinvention is a dsFv fragment of a monoclonal antibody, that is, adisulfide-stabilized Fv fragment in which the heavy chain variableregion and the light chain variable region are linked via a disulfidebond. According to one particular embodiment of the present invention,the dsFv fragment is a heterodimer consisting of a heavy chain variableregion and a light chain variable region linked via a disulfide bond.

In yet another aspect, the present disclosure also provides use of thehumanized anti-CD22 antibody in the preparation of a recombinantimmunotoxin. For example, any chain of the humanized anti-CD22 antibodycan be linked to a cytotoxin of the present invention to prepare arecombinant immunotoxin.

The present invention provides a new humanized anti-CD22 recombinantimmunotoxin composed of a humanized anti-CD22 monoclonal antibodyfragment fused with truncated pseudomonas exotoxin A, specifically aheterodimer composed of a heavy chain variable region and a light chainvariable region linked via a disulfide bond, wherein the heavy chainvariable region is fused with the truncated pseudomonas exotoxin A.

Compared with the recombinant immunotoxin before humanizationengineering, the humanized anti-CD22 recombinant immunotoxin accordingto the present invention is obtained through a specific humanizationstrategy, so better resembles those of human counterparts, and thuspotentially reduce the immunogenicity of the Fv portion of theimmunotoxin in human. The reduced immunogenicity could prevent thegeneration of undesirable neutralizing anti-drug antibodies (ADAs) andcontribute to better pharmacokinetics profiles when used in clinics.

Moreover, the humanized anti-CD22 recombinant immunotoxin according tothe present invention maintains the best affinity for the target CD22and the corresponding biological activity while having maximumhumanization. Compared with the recombinant immunotoxin beforehumanization engineering, the humanized anti-CD22 recombinantimmunotoxin of the present invention maintains the binding affinity forreceptor CD22 and the cell growth inhibitory and apoptotic effects oneffector cells. In addition, the humanized anti-CD22 recombinantimmunotoxin exhibits comparable pharmacokinetics and tumor suppressionefficacy to that one before the engineering.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention are described in detail below withreference to the attached figures, in which:

FIG. 1 shows the schematic diagram of the humanized anti-CD22recombinant immunotoxin according to the present invention.

FIG. 2 shows the alignment results of different versions of humanizedsequences performed in the present disclosure, in which panel 2A: VH;panel 2B: VL.

FIG. 3 shows the expression plasmid maps of the humanized anti-CD22recombinant immunotoxin according to the present invention, in whichpanel 3A: the expression plasmid of the first polypeptide chain; panel3B: the expression plasmid of the second polypeptide chain.

FIG. 4 shows the PK profiles of anti-CD22 recombinant immunotoxins, inwhich panel 4A: HA22; panel 4B: H1L2.

FIG. 5 shows the tumor suppression efficacy of anti-CD22 recombinantimmunotoxins.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As introduced in the present disclosure, a humanization strategy wasemployed to optimize the Fv region of the anti-CD22 portion of HA22 tobetter resemble those of human counterparts, and thus potentially reducethe immunogenicity of the Fv portion of the immunotoxin in human. Thereduced immunogenicity could prevent the generation of undesirableneutralizing anti-drug antibody (ADA) and contribute to betterpharmacokinetics profiles when used in clinics.

Accordingly, the present disclosure provides a humanized anti-CD22recombinant immunotoxin, comprising a humanized anti-CD22 monoclonalantibody fragment fused with truncated pseudomonas exotoxin A.Specifically, the humanized anti-CD22 recombinant immunotoxin is aheterodimer comprised of a kappa light chain V region (a firstpolypeptide chain) and a heavy chain V region linked by one disulfidebond, wherein the heavy chain V region is fused with a truncatedpseudomonas exotoxin A (a second polypeptide chain). The schematicdiagram of the recombinant immunotoxin is shown in FIG. 1 , in which thetruncated pseudomonas exotoxin A is illustratively PE38.

According to the present invention, the humanized anti-CD22 recombinantimmunotoxin binds to CD22 receptor on the malignant B-cell surface todeliver the toxin moiety e.g., PE38 directly to tumor cells. Uponinternalization, the toxin part catalyzes the ADP ribosylation of thediphthamide residue in elongation factor-2 (EF-2), resulting in therapid fall in levels of the anti-apoptotic protein myeloid cell leukemia1 (Mcl-1), leading to apoptotic cell death. The humanized anti-CD22recombinant immunotoxin according to the present invention can beproduced in, for example, an enhanced BL21 derivative strain (T7Express) containing an expression plasmid harboring nucleotide sequencecoding for the first polypeptide chain and an expression plasmidharboring nucleotide sequence coding for the second polypeptide chain.

The resulting strain is resistant to kanamycin and the production of thehumanized anti-CD22 recombinant immunotoxin is induced by Isopropylβ-d-1-thiogalactopyranoside (IPTG). The product is expressed asinclusion bodies. The inclusion bodies can be recovered and thenrefolded in vitro to form a heterodimer. The refolded protein can bethen purified by hydrophobic interaction chromatography followed byanion exchange chromatography. The purified protein is finallyconcentrated and diafiltered into a buffer prepared for storage.

The humanized anti-CD22 recombinant immunotoxin according to the presentinvention is a humanized version of HA22, and therefore is expected tointeract with CD22 expressing cells via identical/similar mechanism ofaction(s). In vitro and in vivo assays were designed to assessimmunotoxin molecules' CD22 binding affinity, cytotoxicity inCD22-expressing cells, pharmacokinetics and tumor suppression efficacy.HA22 used in the studies was manufactured in-house using the sameproduction process as that of the recombinant immunotoxin.

In the present disclosure, the interaction between the antibody fragmentof the humanized anti-CD22 recombinant immunotoxin of the presentinvention and the CD22 receptor was determined in in vitro bindingassay; and the growth inhibition and apoptotic cell death effects of thehumanized anti-CD22 recombinant immunotoxin were examined in cell-basedassay. Further, the pharmacokinetic and tumor suppression efficacy ofthe humanized anti-CD22 recombinant immunotoxin of the present inventionwere determined in in vivo studies performed.

Sequences used in the present disclosure are as follows:

SEQ ID NO. 1: PE38 polypeptide sequenceKASGGP EGGSLAALTA HQACHLPLET FTRHRQPRGW EQLEQCGYPV QRLVALYLAARLSWNQVDQV IRNALASPGS GGDLGEAIRE QPEQARLALT LAAAESERFVRQGTGNDEAG AANGPADSGD ALLERNYPTG AEFLGDGGDV SFSTRGTQNWTVERLLQAHR QLEERGYVFV GYHGTFLEAA QSIVEGGVRA RSQDLDAIWRGFYIAGDPAL AYGYAQDQEP DARGRIRNGA LLRVYVPRSS LPGFYRTSLTLAAPEAAGEV ERLIGHPLPL RLDAITGPEE EGGRLETILG WPLAERTVVIPSAIPTDPRN VGGDLDPSSI PDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 2: HA22, VH polypeptide sequence (HA22 V_(H))MEVQLVESGG GLVKPGGSLK LSCAASGFAF SIYDMSWVRQ TPEKCLEWVAYISSGGGTTY YPDTVKGRFT ISRDNAKNTL YLQMSSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTLV TVSASEQ ID NO. 3: Version 1 VH polypeptide sequence (V_(H) ver 1)MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ APGKCLEWVAYISSGGGTTY YPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTMV TVSSSEQ ID NO. 4: Version 2 VH polypeptide sequence (V_(H) ver 2)MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ TPEKCLEWVAYISSGGGTTY YPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTMV TVSSSEQ ID NO. 5: Version 3 VH polypeptide sequence (V_(H) ver 3)MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVSYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSSEQ ID NO. 6: Version 4 VH polypeptide sequence (V_(H) ver 4)MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVSYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSSEQ ID NO. 7: Version 5 VH polypeptide sequence (V_(H) ver 5)MEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVAYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRSE DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSSEQ ID NO. 8: HA22 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLK LSCAASGFAF SIYDMSWVRQ TPEKCLEWVAYISSGGGTTY YPDTVKGRFT ISRDNAKNTL YLQMSSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTLV TVSAKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVEGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 9: Version 1 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ APGKCLEWVAYISSGGGTTY YPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTMV TVSSKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVEGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 10: Version 2 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWVRQ TPEKCLEWVAYISSGGGTTY YPDSVKGRFT ISRENAKNSL YLQMNSLKSE DTAMYYCARHSGYGTHWGVL FAYWGQGTMV TVSSKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 11: Version 3 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVSYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 12: Version 4 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVAYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRAG DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGIGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVEGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 13: Version 5 VH-PE38 polypeptide sequenceMEVQLVESGG GLVKPGGSLR LSCAASGFAF SIYDMSWIRQ APGKCLEWVAYISSGGGTTY YPGSVKGRFT ISRENAKNSL YLQMNSLRSE DTAVYYCARHSGYGTHWGVL FAYWGQGTMV TVSSKASGGP EGGSLAALTA HQACHLPLETFTRHRQPRGW EQLEQCGYPV QRLVALYLAA RLSWNQVDQV IRNALASPGSGGDLGEAIRE QPEQARLALT LAAAESERFV RQGTGNDEAG AANGPADSGDALLERNYPTG AEFLGDGGDV SFSTRGTQNW TVERLLQAHR QLEERGYVFVGYHGTFLEAA QSIVFGGVRA RSQDLDAIWR GFYIAGDPAL AYGYAQDQEPDARGRIRNGA LLRVYVPRSS LPGFYRTSLT LAAPEAAGEV ERLIGHPLPLRLDAITGPEE EGGRLETILG WPLAERTVVI PSAIPTDPRN VGGDLDPSSIPDKEQAISAL PDYASQPGKP PREDLKSEQ ID NO. 14: HA22, VL polypeptide sequence (HA22 V_(L))MDIQMTQTTS SLSASLGDRV TISCRASQDI SNYLNWYQQK PDGTVKLLIYYTSILHSGVP SRFSGSGSGT DYSLTISNLE QEDFATYFCQ QGNTLPWTFG CGTKLEIKSEQ ID NO. 15: Version 1 VL polypeptide sequence (V_(L) ver 1)MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWYQQR PGQSVKLLIYYTSILHSGVP SRFSGSGSGT DYTLTISSLQ PEDFATYFCQ QGNTLPWTFG CGTKVEIKSEQ ID NO. 16: Version 2 VL polypeptide sequence (V_(L) ver 2)MDIQMTQSTS TLSASVGDRV TITCRASQDI SNYLNWYQQR PDGSVKLLIYYTSILHSGVP SRFSGSGSGT DYTLTISSLQ DEDFATYFCQ QGNTLPWTFG CGTKVEIKSEQ ID NO. 17: Version 3 VL polypeptide sequence (V_(L) ver 3)MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWFQQR PGQSPRRLIYYTSILHSGVP SRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG CGTKVEIKSEQ ID NO. 18: Version 4 VL polypeptide sequence (V_(L) ver 4)MDIQMTQSPS TLSASVGDRV TITCRASQDI SNYLNWFQQR PGQSPRLLIYYTSILHSGVP SRFSGSGSGT DYTLTISSLQ PEDFATYYCQ QGNTLPWTFG  CGTKVEIKSEQ ID NO. 19: Version 1 VH-PE38 DNA sequenceATGGAAGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCAAGCCCGGCGGCTCTCTGAGACTGTCCTGCGCTGCTTCTGGCTTCGCCTTCTCCATCTACGACATGTCCTGGGTCAGACAGGCCCCTGGCAAGTGCCTGGAATGGGTGGCCTACATCTCCTCTGGCGGAGGCACCACCTACTACCCTGACTCCGTGAAGGGCAGATTCACCATCTCTAGAGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAGTCCGAGGACACCGCCATGTACTACTGCGCCAGACACTCCGGCTACGGCACACACTGGGGCGTGCTGTTCGCTTACTGGGGCCAGGGCACCATGGTGACCGTGTCCTCCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAASEQ ID NO. 20: Version 2 VH-PE38 DNA sequenceATGGAAGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCAAGCCCGGCGGCTCTCTGAGACTGTCCTGCGCTGCTTCTGGCTTCGCCTTCTCCATCTACGACATGTCCTGGGTCAGACAGACCCCTGAAAAGTGCCTGGAATGGGTGGCCTACATCTCCTCTGGCGGAGGCACCACCTACTACCCTGACTCCGTGAAGGGCAGATTCACCATCTCTAGAGAGAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAGTCCGAGGACACCGCCATGTACTACTGCGCCAGACACTCCGGCTACGGCACACACTGGGGCGTGCTGTTCGCTTACTGGGGCCAGGGCACCATGGTGACCGTGTCCTCCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAASEQ ID NO. 21: Version 3 VH-PE38 DNA sequenceATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGAGCTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTGCGGGTGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA SEQ ID NO. 22: Version 4 VH-PE38 DNA sequenceATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGGCGTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTGCGGGTGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA SEQ ID NO. 23: Version 5 VH-PE38 DNA sequenceATGGAAGTGCAGCTGGTTGAAAGCGGTGGCGGTCTGGTGAAACCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTTCGCGTTTAGCATCTACGACATGAGCTGGATTCGTCAAGCGCCGGGCAAATGCCTGGAGTGGGTGGCGTATATCAGCAGCGGCGGTGGCACCACCTACTATCCGGGTAGCGTTAAGGGCCGTTTCACCATTAGCCGTGAAAACGCGAAAAACAGCCTGTACCTGCAGATGAACAGCCTGCGTTCTGAGGATACCGCTGTGTACTATTGCGCGCGTCACAGCGGTTACGGCACCCACTGGGGCGTTCTGTTTGCGTATTGGGGTCAAGGCACCATGGTGACCGTTAGCAGCAAAGCGTCAGGCGGTCCGGAAGGCGGTTCGCTGGCAGCTCTGACCGCACATCAGGCATGCCACCTGCCGCTGGAAACCTTTACCCGCCATCGTCAACCGCGCGGCTGGGAACAGCTGGAACAATGTGGTTATCCGGTGCAGCGCCTGGTTGCCCTGTACCTGGCCGCACGTCTGAGCTGGAACCAGGTTGATCAAGTCATTCGTAATGCGCTGGCATCACCGGGCTCGGGCGGTGACCTGGGTGAAGCAATCCGCGAACAGCCGGAACAAGCGCGCCTGGCCCTGACCCTGGCAGCTGCGGAATCCGAACGCTTTGTGCGTCAGGGCACCGGTAATGATGAAGCCGGTGCAGCAAATGGTCCGGCTGATTCAGGTGACGCGCTGCTGGAACGCAACTATCCGACGGGCGCCGAATTTCTGGGTGATGGCGGTGACGTGAGTTTCTCCACCCGCGGCACGCAGAATTGGACCGTTGAACGTCTGCTGCAGGCGCATCGCCAACTGGAAGAACGTGGTTATGTTTTTGTCGGCTATCATGGCACCTTTCTGGAAGCTGCGCAGTCGATTGTCTTTGGCGGTGTGCGTGCACGCAGCCAGGATCTGGATGCAATTTGGCGCGGCTTCTACATCGCAGGTGATCCGGCTCTGGCGTATGGCTACGCTCAGGATCAAGAACCGGATGCGCGTGGCCGCATCCGTAATGGTGCCCTGCTGCGTGTGTATGTTCCGCGTTCATCGCTGCCGGGTTTTTACCGTACCTCTCTGACCCTGGCGGCACCGGAAGCTGCCGGCGAAGTGGAACGCCTGATTGGTCACCCGCTGCCGCTGCGTCTGGATGCAATCACCGGTCCGGAAGAAGAAGGCGGCCGTCTGGAAACGATTCTGGGTTGGCCGCTGGCCGAACGTACCGTGGTTATTCCGAGCGCAATCCCGACGGACCCGCGCAATGTTGGCGGTGATCTGGACCCGAGCTCTATTCCGGATAAAGAACAGGCCATCTCCGCACTGCCGGACTATGCGTCACAACCGGGCAAACCGCCGCGTGAAGACCTGAAA SEQ ID NO. 24: Version 1 VL DNA sequenceATGGACATCCAGATGACCCAGTCTCCTTCTACACTGTCTGCTTCTGTGGGCGACAGAGTGACCATCACCTGCAGAGCCTCTCAGGACATCTCCAACTACCTGAACTGGTACCAGCAGAGACCTGGCCAGTCCGTGAAGCTGCTGATCTACTACACCTCCATCCTGCACTCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGATCTGGCACCGACTACACCCTGACCATCTCCTCCCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCTTGGACCTTCGGCTGCGGCACCAAGGTGGAAATCAAGSEQ ID NO. 25: Version 2 VL DNA sequenceATGGACATCCAGATGACCCAGTCTACTTCTACACTGTCTGCTTCTGTGGGCGACAGAGTGACCATCACCTGCAGAGCCTCTCAGGACATCTCCAACTACCTGAACTGGTACCAGCAGAGACCTGATGGCTCCGTGAAGCTGCTGATCTACTACACCTCCATCCTGCACTCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGATCTGGCACCGACTACACCCTGACCATCTCCTCCCTGCAGGATGAGGACTTCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCTTGGACCTTCGGCTGCGGCACCAAGGTGGAAATCAAGSEQ ID NO. 26: Version 3 VL DNA sequenceATGGACATCCAGATGACCCAGAGCCCGAGCACCCTGAGCGCGAGCGTGGGCGACCGTGTTACCATCACCTGCCGTGCGAGCCAGGATATTAGCAACTACCTGAACTGGTTTCAACAACGTCCGGGTCAAAGCCCGCGTCGTCTGATCTACTATACCAGCATTCTGCACAGCGGTGTGCCGAGCCGTTTTAGCGGTAGCGGCAGCGGTACCGACTATACCCTGACCATCAGCAGCCTGCAGCCGGAGGATTTCGCGACCTACTATTGCCAGCAGGGTAACACCCTGCCGTGGACCTTTGGCTGCGGTACCAAGGTTGAAATTAAASEQ ID NO. 27: Version 3 VL DNA sequenceATGGACATCCAGATGACCCAGAGCCCGAGCACCCTGAGCGCGAGCGTGGGCGACCGTGTTACCATCACCTGCCGTGCGAGCCAGGATATTAGCAACTACCTGAACTGGTTTCAACAACGTCCGGGTCAAAGCCCGCGTctgCTGATCTACTATACCAGCATTCTGCACAGCGGTGTGCCGAGCCGTTTTAGCGGTAGCGGCAGCGGTACCGACTATACCCTGACCATCAGCAGCCTGCAGCCGGAGGATTTCGCGACCTACTATTGCCAGCAGGGTAACACCCTGCCGTGGACCTTTGGCTGCGGTACCAAGGTTGAAATTAAA SEQ ID NO. 28: HA22, H-CDR1GFAFSIYD SEQ ID NO. 29: HA22, H-CDR2 ISSGGGTTYSEQ ID NO. 30: HA22, H-CDR3 CARHSGYGTHWGVLFAYSEQ ID NO. 31: HA22, L-CDR1 QDISNY SEQ ID NO. 32: HA22, L-CDR2 YTSILHSGSEQ ID NO. 33: HA22, L-CDR3 QQGNTLP

The present invention is illustrated below with reference to specificexamples. It will be understood by those skilled in the art that theseexamples are merely illustrative of the present invention and do notlimit the scope of the present invention in any way.

Experimental procedures in the following examples are all conventionalones, unless otherwise specified. Raw materials and reagents used in thefollowing examples are all commercially available products, unlessotherwise specified.

Example 1: Humanization Design

A framework shuffling strategy was used to alter amino acids in theframework regions of heavy chain variable region (V_(H), SEQ ID NO. 2)and light chain variable region (V_(L), SEQ ID NO. 14) from the originalmurine to human ones. All the CDRs, which were determined using both theKabat and Chothia numbering systems and the online tool(http://www.bioinf.org.uk/abs/), were left unchanged. Each of theframework and J regions in both the VH and VL of HA22 was aligned to thehuman antibody germline sequences in the international ImMunoGeneTicsinformation system® (IMGT: http://www.imgt.org) for the closest humanantibody matches. Murine residues that were deemed Vernier residues orimportant for VH/VL interactions were not changed^([26]) to preserve thespecificity and affinity towards CD22.

A panel of humanization designs were prepared and evaluated to selectthe final candidate that possessed maximum humanization whilemaintaining optimal affinity and efficacy profile. The updated VH and VLformed a disulfide-linked Fv portion of anti-CD22 antibody fused to aPseudomonas exotoxin A (PE38, SEQ ID NO. 1) (FIG. 1 ).

The alignment revealed a combination of human germline sequencesframework 1 of IGHV3-15*01, framework 2 of IGHV3-11*04, framework 3 ofIGHV3-13*01, and framework 4 of IGHJ3*02 would provide humanizedframeworks most closely resembling those of VH of HA22; and similarly acombination of framework 1 of IGKV1-5*01, framework 2 of IGKV2D-30*01,framework 3 of IGKV1D-43*01, and framework 4 of IGKJ1*01 would providehumanized frameworks most closely resembling those of VL of HA22. TheVernier residues were reverted to the original murine residues afterrounds of iterations to show that they were critical for antigenbindings.

The sequence alignment provided in FIG. 2 shows examples of thehumanization process. In the alignment, HA22VH and HA22VL are the Fvfragments used in HA22. Version 3 (V3) is the fully humanized VH and VLversions. Version 1, 2, 4, and 5 (V1, V2, V4, and V5) are differentversions of designs that have critical residues reverted to murine onesto restore lost activities. Residues shaded are Vernier residues.Residues underlined are in CDRs based on IMGT criteria.

Example 2: Production of the Humanized Anti-CD22 Recombinant Immunotoxin

Expression plasmids capable of expressing different VL humanizedversions (the first polypeptide chains) were constructed. In addition,5′ terminus of the nucleotide sequence coding for PE38 was linked to 3′terminus of the nucleotide sequence coding for each of the different VHhumanized versions respectively to form a single nucleotide chain, withwhich expression plasmids capable of expressing the second polypeptidechains were constructed. Schematic diagrams are shown in FIG. 3(synthesized and constructed by Genscript according to the designs).

The humanized anti-CD22 recombinant immunotoxins provided in the presentdisclosure were produced in an enhanced BL21 derivative strain (T7Express) using the expression plasmids of the first and secondpolypeptide chains.

1. Fermentation

The first polypeptide chain and second polypeptide chain seed cultureswere prepared, respectively, in a fermentation medium containing 30 gsoytone, 30 g yeast extract, 40 g glycerol, 2 g NH₄Cl, 2 g (NH₄)₂SO₄,0.973 g MgSO₄, 3 g glucose, and 1 g NaCl per L. The seed cultures wereincubated at 37° C., 250 RPM for 12-16 hours. The fermentation runs forthe first polypeptide chain and second polypeptide chain were performedusing a New Brunswick BioFlo 3000 Fermentor. The fermentor wasinoculated at 2% with the respective first polypeptide chain and secondpolypeptide chain seed. The pH was set at 6.9 and controlled during therun by addition of ammonia (NH₃⋅H₂O). The dissolved oxygen (DO) was setat 30% and controlled by agitation, aeration and oxygen supplementation.The vessel pressure was set at 2 psi. The cell density (OD600) andglucose concentrations, together with pH, DO, and temperature, weremonitored hourly during the run. Four hours post inoculation, at OD6008-9, the glucose in the medium was depleted and additional glucose wasfed into the fermentor. The glucose feeding lasted for an hour, with atotal volume of 1.7% of original medium volume. The additional glucosewas exhausted at around 5.5 hours after inoculation. 0.1% volume of 1 MIPTG was then added to start the induction/expression phase. During theinduction stage, pH was allowed to increase gradually to 7.0. Threehours post induction, the cells were harvested by centrifugation at8,000×g, at 4° C., for 20 minutes to remove the medium. The cell pastewas stored at −80° C. before processing.

2. Recovery and Refolding

The first polypeptide chain and second polypeptide chain inclusionbodies were isolated by disrupting the cells using a high-pressurehomogenizer. The lysate pellet containing the inclusion body was thenpurified through several washing steps with centrifugation and detergentto remove cell debris and soluble impurities.

The washed inclusion bodies were resuspended in a solubilization buffer(50 mM ethanolamine, 8 M Urea, 0.5 M arginine, 2 mM EDTA, 10 mM DTT, pH9.3) containing a strong denaturant (8 M urea) and a reducing agent (10mM DTT) to keep all cysteines in the reduced state and cleave disulfidebonds formed during the preparation. Solubilization was performed at2-8° C. with stirring to facilitate the solubilization process for >2hours. After solubilization, the inclusion body solution was centrifugedat 13,000×g for 30 minutes and the supernatant containing thesolubilized inclusion body was filtered to remove the remainingaggregates and insoluble impurities.

Total protein concentrations were determined based on A280 reading andpercentages of the first polypeptide chain and second polypeptide chainwere estimated by densitometry analysis of SDS-PAGE gel. The firstpolypeptide chain and second polypeptide chain were then mixed at 1:1molar ratio, diluted to 10 mg/mL with solubilization buffer, and fedinto the refolding buffer (50 mM ethanolamine, 0.5 M arginine, 2 mMEDTA, 0.9 mM oxidized glutathione, pH 9.4) at a 1:10 ratio continuouslyover a 24-hour period and incubated at 2-8° C. for an additional 48-72hours. The refolding reaction was stopped by adjusting the pH to ˜7.40.The refolded protein was then concentrated and prepared for columnpurification by tangential flow filtration.

3. Phenyl HP Hydrophobic Interaction Chromatography

Phenyl HP hydrophobic interaction chromatography was carried out in achromatography column with 17 cm bench height (GE Healthcare,Marlborough, MA USA). All runs were conducted using an AKTA avant liquidchromatography system from GE Healthcare (Marlborough, MA USA) and thecolumn was operated at 95 cm/hr. The column was equilibrated with 20 mMTris-HCl, 0.6 M Na₂SO₄, pH 7.4. Load was prepared by diluting 1 part (byweight) protein solution with 1 part 20 mM Tris-HCl, 1.2 M Na₂SO₄, pH7.4. After loading, the column was washed with equilibration buffer andthen eluted in a linear gradient of Na₂SO₄ from 0.6 M to 0 M over 16column volumes. The product peak was collected in fractions.

4. POROS HQ Anion Exchange Chromatography

POROS HQ anion exchange chromatography was carried out in achromatography column packed to 30 cm bed height (Thermo Scientific,Waltham, MA USA). All runs were conducted using an AKTA avant liquidchromatography system from GE Healthcare and the column was operated at275 cm/hr. The column was equilibrated with 20 mM Tris-HCl, pH 7.4,loaded with protein, and then washed with equilibration buffer. Thecolumn was eluted in a linear gradient of NaCl from 0 M to 0.5 M over 25column volumes. The product peak was collected based on absorbancecriteria of 5 mAU on the leading and tailing side of the product peak.

5. Capto Q Anion Exchange Chromatography

Capto Q anion exchange chromatography was carried out in flow-throughmode in a chromatography column packed to 10 cm bed height (GEHealthcare, Marlborough, MA USA). All runs were conducted using an AKTAavant liquid chromatography system from GE Healthcare and the column wasoperated at 250-350 cm/hr. The column was equilibrated with 20 mMTris-HCl, 250 mM NaCl, pH 7.4, loaded with protein, and then washed withequilibration buffer. The product peak was collected based on absorbancecriteria of 5 mAU on the leading and tailing side of the productflow-through peak.

Example 3: Non-Clinical Pharmacology of the Humanized Anti-CD22Recombinant Immunotoxins

As described in Example 2, different recombinant immunotoxins wereprepared with different VH and VL humanized versions provided in thepresent disclosure, as well as with the VH and VL of HA22, and werenamed following a format “HmLn”, in which m and n is the number of theVH and VL humanized versions.

3.1 Target Interaction

ELISA was performed with recombinant human Siglec-2/CD22 Fc chimeraprotein (R&D systems, Cat #1968-SL) as the immobilized antigen, whichcaptures the humanized anti-CD22 recombinant immunotoxins or HA22molecule provided in the present disclosure, and anti-PseudomonasExotoxin A antibody (Sigma-Aldrich, Cat #P2318) as the secondaryantibody containing the enzyme conjugate for detection.

Octet assay was performed with streptavidin (SA) biosensors (ForteBio,Cat #18-5019) conjugated with biotinylated CD22 for the K_(D)measurement of the recombinant immunotoxins provided in the presentdisclosure or HA22 molecule.

As shown in Table 1, recombinant immunotoxins H1L2 (containing V_(H) ver1+V_(L) ver 2) and H2L2 (containing V_(H) ver 2+V_(L) ver 2) showedcomparable EC₅₀ and K_(D) to HA22.

TABLE 1 CD22 binding assay results Sample EC₅₀ by ELISA K_(D) by OctetHA22 0.47 ± 0.48 nM 4.08-5.81 nM H1L2 0.54 ± 0.24 nM 4.66-7.21 nM H2L20.56 ± 0.33 nM 5.01-7.02 nM

Different versions of the humanized recombinant immunotoxins wereanalyzed for CD22 binding affinity, as shown in Table 2 and Table 3. InTable 2, a range of EC₅₀ by ELISA is given or presented as Mean±SD whentwo or no less than three batches of products were assayed in separateanalyses, respectively. Results of EC₅₀ of the humanized recombinantimmunotoxins relative to HA22 are shown in Table 3.

TABLE 2 CD22 binding affinity (nM) →Decreasing numbers of humanizationchanges→ V_(H) ver 3 V_(H) ver 4 V_(H) ver 5 V_(H) ver 1 V_(H) ver 2HA22 V_(H) →Decreasing V_(L) ver 3 ND ND numbers of V_(L) ver 4 6.732.15 0.2 humanization V_(L) ver 1 V_(L) ver 1, no expression changes→V_(L) ver 2 0.54 ± 0.24 0.56 ± 0.33 HA22 V_(L) 0.64 ± 0.12 0.47 ± 0.48

TABLE 3 CD22 binding affinity (EC₅₀, normalized against HA22)→Decreasing numbers of humanization changes→ V_(H) ver 3 V_(H) ver 4V_(H) ver 5 V_(H) ver 1 V_(H) ver 2 HA22 V_(H) →Decreasing V_(L) ver3 >>1000 >>1000     numbers of V_(L) ver 4 39.07 12.49  1.17humanization V_(L) ver 1 No expression changes→ V_(L) ver 2 0.81 ± 0.181.1 HA22 V_(L) 0.89 ± 0.05 1

3.2 Cell-Based Potency Assay

A CD22-expressing cell model was employed in cell-based assay toevaluate the growth inhibition and apoptotic cell death effects of thehumanization anti-CD22 recombinant immunotoxins in the presentdisclosure. This is an endpoint assay using cell growth/inhibition asthe indicator of potency. The cell model better mimics the in vivocondition comparing to the binding assay, in which, the CD22 binding,internalization, and ADP ribosylation of EF-2 catalyzed by PE38 are allinvolved to actuate the observed inhibitory effect.

In this assay, the Daudi cell line expressing a high level of CD22, wasco-cultured with the recombinant immunotoxins or HA22 molecule providedin the present disclosure for a certain period of time and, at the endof the assay, live cells in the culture were quantified with acolorimetric assay. The procedure is as follows.

Daudi cells described above were cultured in RPMI-1640 complete medium,and harvested and washed with HBSS once. The cells were resuspended inRPMI-1640 medium at a density of 2.5×10⁵/mL, and transferred to a96-well plate at 100 μL/well (equivalent to 2.5×10⁴ live cells perwell). The recombinant immunotoxins or HA22 provided in the presentdisclosure to be detected were diluted to predetermined concentrationsand serial dilutions were prepared.

100 μL of each dilution was added to the cells in the wells, and thecells were incubated at 37° C., 5% CO₂ for 48-72 hours. Afterwards, 10μL of CCK-8 agent was added into each well of the plate which was thenincubated at 37° C. for 4-8 hours. To correct for background activity,the cells were cultured in the presence of 10 μg/mL cycloheximide until100% of the cells died.

The generation of Formazan was measured by detecting the absorbance at450 nm. Values were normalized against cycloheximide and medium control,and the concentration of each of the humanized recombinant immunotoxinsand HA22 provided in the present disclosure when 50% of the cells diedwas measured as EC₅₀.

Different versions of the humanized recombinant immunotoxins wereanalyzed for CD22 binding affinity, as shown in Table 4 and Table 5. InTable 4, a range of EC₅₀ is given or presented as Mean f SD when two orno less than three batches of products were assayed in separateanalyses, respectively. Results of EC₅₀ of the humanized recombinantimmunotoxins relative to HA22 are shown in Table 5. As shown, thehumanized recombinant immunotoxins, especially H1L2 showed comparableEC₅₀ to HA22 in in vitro cell-based potency assay.

TABLE 4 In Vitro potency assay (pM) →Decreasing numbers of humanizationchanges→ V_(H) ver 3 V_(H) ver 4 V_(H) ver 5 V_(H) ver 1 V_(H) ver 2HA22 V_(H) →Decreasing V_(L) ver 3 ND 57.16 numbers of V_(L) ver 4 ND34.85 ± 0.12 13.83 ± 0.38 V_(L) ver 1 No expression V_(L) ver 2  4.58 ±1.57 4.26 ± 1.59 HA22 V_(L) 2.72 ± 0.55

TABLE 5 In Vitro potency assay (EC₅₀, normalized against HA22)→Decreasing numbers of humanization changes→ V_(H) ver 3 V_(H) ver 4V_(H) ver 5 V_(H) ver 1 V_(H) ver 2 HA22 V_(H) →Decreasing V_(L) ver 3ND 4.59 numbers of V_(L) ver 4 ND 10.7 4.12 humanization V_(L) ver 1V_(L) ver 1, no expression changes→ V_(L) ver 2 1.32 ± 0.17 1.23 HA22V_(L) 1

Example 4: Non-Clinical Pharmacokinetics and Drug Metabolism of theHumanized Anti-CD22 Recombinant Immunotoxins

4.1 In Vivo PK Studies

A C57BL/6 mouse model (4-6 week-old, female) was used in this study. 45mice were randomly divided into 3 groups: 1) control (formulationbuffer), 2) 500 μg/kg HA22; and 3) 500 μg/kg H1L2. The control ortreatment groups were given via tail vein injection, and 100 μL totalblood was collected at specified time points after injection (5 min, 15min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 96 h) via retroorbital sampling. Three replicate animals were assigned to each timepoint. 50 μL plasma was recovered from the blood sample for analysis.

As shown in Table 6 and Table 7, results showed that in the mouse modelH1L2 showed comparable pharmacokinetics properties to those of HA22:both molecules exhibited similar half-life (T_(1/2)), time to reachmaximum concentration (T_(max)), maximum concentration (C_(max)) as wellas area under the curve (AUC₀₋₄). PK profile is shown in FIG. 4 .

TABLE 6 HA22 PK result AUC_(0-t) T_(1/2) (min) T_(max) (min) C_(max)(ng/mL) (ng/mL × min) Replicate 1 44.38 5 1.671 × 10⁴ 7.329 × 10⁵Replicate 2 46.32 5 1.275 × 10⁴ 6.385 × 10⁵ Replicate 3 57.26 5 1.863 ×10⁴ 7.125 × 10⁵ Average 49.32 5 1.603 × 10⁴ 6.947 × 10⁵

TABLE 7 H1L2 PK result AUC_(0-t) T_(1/2) (min) T_(max) (min) C_(max)(ng/ml) (ng/ml × min) Replicate 1 41.69 5 1.906 × 10⁴ 7.416 × 10⁵Replicate 2 41.76 5 1.581 × 10⁴ 7.458 × 10⁵ Replicate 3 42.48 5 1.910 ×10⁴ 7.474 × 10⁵ Average 41.98 5 1.799 × 10⁴ 7.449 × 10⁵

4.2 In Vivo Efficacy

A xenograft animal model and the C1D22-positive Raji B lymphocytes wereemployed for tumor establishment. Upon administration, the recombinantimmunotoxin molecule enters the circulation, binds to the CD22 on thesurface of the malignant B-cells, and suppresses the cell growth via theapoptotic pathways. Therefore, the inhibition of tumor growth wasdesignated as the endpoint of the efficacy study in this Example.

An athymic NCr nude mouse model (4-6 week-old, female) was used. 25 micewere randomly divided into 5 groups as shown in Table 8. A CD-22positive Homo sapiens lymphoblast Raji cell line (ATCC CCL-86) was usedto establish the xenograft. Raji cells were subcutaneously injected at afinal concentration of 5×10⁷ cells/mL and grown to 100-200 mm³ beforeany treatment. Once the xenograft tumor was established, a single doseof the treatments at the indicated dose level was given via tail veininjection. The xenograft mice were observed and measured for tumor sizefor 24 days after the treatment.

As shown in FIG. 5 , comparing to the control group (the first group),HA22 high dose group showed 84.9% suppression of tumor growth, low dosegroup showed 95.8% suppression of tumor growth; and, H1L2 high dosegroup showed 87.7% suppression of tumor growth, low dose group showed34.2% suppression of tumor growth. A T test was performed, and alltreatments except for the humanized recombinant immunotoxin H1L2 at lowdose showed significant tumor suppression efficacy (in FIG. 5 , *P<0.05, ** P<0.01, *** P<0.005). There were no statistic differencesbetween the high dose groups of HA22 and H1L2.

TABLE 8 Efficacy study group and dosing information Group Treatment Doselevel (μg/kg) No. of animals 1 Formulation buffer 0 5 2 HA22 100 5 3HA22 300 5 4 H1L2 100 5 5 H1L2 300 5

The above description of the embodiments of the present invention is notintended to limit the present invention, and those skilled in the artmay make various changes and modifications to the present inventionwithout departing from the spirit of the present invention, which shouldfall within the scope of the appended claims.

REFERENCES

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1. A humanized anti-CD22 recombinant immunotoxin which is a polypeptidemolecule comprising two polypeptide chains as follows: (1) a firstpolypeptide chain, comprising a light chain variable region (VL) of ananti-CD22 antibody, wherein the light chain variable region (VL)comprises an amino acid sequence as shown in SEQ ID NO. 16 or SEQ ID NO.18 or an amino acid sequence homologue thereof having at least 75%sequence identity to the amino acid sequence as shown in SEQ ID NO. 16or SEQ ID NO. 18; (2) a second polypeptide chain, comprising a heavychain variable region (V_(H)) of an anti-CD22 antibody and a cytotoxindirectly or indirectly linked to the heavy chain variable region(V_(H)), wherein the heavy chain variable region (V_(H)) comprises anamino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 or an aminoacid sequence homologue thereof having at least 75% identity to theamino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO.
 4. 2. Thehumanized anti-CD22 recombinant immunotoxin according to claim 1,wherein the amino acid sequence homologue in the first polypeptide chainhas at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to the amino acidsequence as shown in SEQ ID NO. 16 or SEQ ID NO. 18; preferably, theamino acid sequence homologue comprises the amino acid sequences asshown in SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33, and has thesame amino acid residues as those in SEQ ID NO. 16 or SEQ ID NO. 18 atpositions 3, 5, 36, 37, 47, 48, 49, 50, 65, 67, 69, 70 and 72corresponding to the amino acid sequence as shown in SEQ ID NO. 14, butis not the amino acid sequence as shown in SEQ ID NO. 14; preferably,the amino acid sequence homologue in the second polypeptide chain has atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceas shown in SEQ ID NO. 3 or SEQ ID NO. 4; preferably, the amino acidsequence homologue comprises the amino acid sequences as shown in SEQ IDNO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, and has the same amino acidresidues as those in SEQ ID NO. 3 or SEQ ID NO. 4 at positions 3, 48,49, 50, 69, 71, 73, 75, and 80 corresponding to the amino acid sequenceas shown in SEQ ID NO. 2, but is not the amino acid sequence as shown inSEQ ID NO.
 2. 3. The humanized anti-CD22 recombinant immunotoxinaccording to claim 1, wherein the light chain variable region (V_(L)) inthe first polypeptide chain comprises the amino acid sequence as shownin SEQ ID NO. 16 or the amino acid sequence homologue thereof; and theheavy chain variable region (V_(H)) in the second polypeptide chaincomprises the amino acid sequence as shown in SEQ ID NO. 3 or the aminoacid sequence homologue thereof; or the light chain variable region(V_(L)) in the first polypeptide chain comprises the amino acid sequenceas shown in SEQ ID NO. 18 or the amino acid sequence homologue thereof;and the heavy chain variable region (VH) in the second polypeptide chaincomprises the amino acid sequence as shown in SEQ ID NO. 3 or the aminoacid sequence homologue thereof; or the light chain variable region(V_(L)) in the first polypeptide chain comprises the amino acid sequenceas shown in SEQ ID NO. 16 or the amino acid sequence homologue thereof;and the heavy chain variable region (V_(H)) in the second polypeptidechain comprises the amino acid sequence as shown in SEQ ID NO. 4 or theamino acid sequence homologue thereof; preferably, in the recombinantimmunotoxin, the light chain variable region (V_(L)) in the firstpolypeptide chain and the heavy chain variable region (V_(H)) in thesecond polypeptide chain are covalently linked, e.g., via a disulfidebond; preferably, the cytotoxin in the second polypeptide chain ispseudomonas exotoxin A or a mutant or fragment of the pseudomonasexotoxin A with cytotoxicity retained; preferably, the mutant orfragment of the pseudomonas exotoxin A is PE40, PE38, PE35, PE24, mPE24,T19, T20, or M11.
 4. The humanized anti-CD22 recombinant immunotoxinaccording to claim 1, wherein in the second polypeptide chain, thecytotoxin is fused to the C-terminus of the heavy chain variable region(V_(H)) of the anti-CD22 antibody; preferably, the N-terminus of thecytotoxin is fused, directly or via a linker, to the C-terminus of theheavy chain variable region (V_(H)); preferably, the first polypeptidechain further comprises a kappa light chain constant region, preferablya human kappa light chain constant region fused to the C-terminus of thelight chain variable region (V_(L)); alternatively, the secondpolypeptide chain further comprises a heavy chain constant region CH1,preferably a human heavy chain constant region CH1 fused to theC-terminus of the heavy chain variable region (V_(H)).
 5. The humanizedanti-CD22 recombinant immunotoxin according to claim 1, wherein thefirst polypeptide chain comprises the amino acid sequence as shown inSEQ ID NO. 16 or the amino acid sequence homologue thereof, and thesecond polypeptide chain comprises the amino acid sequence as shown inSEQ ID NO. 9 or the amino acid sequence homolog thereof; or the firstpolypeptide chain comprises the amino acid sequence as shown in SEQ IDNO. 18 or the amino acid sequence homologue thereof, and the secondpolypeptide chain comprises the amino acid sequence as shown in SEQ IDNO. 9 or the amino acid sequence homologue thereof; or the firstpolypeptide chain comprises the amino acid sequence as shown in SEQ IDNO. 16 or the amino acid sequence homologue thereof, and the secondpolypeptide chain comprises the amino acid sequence as shown in SEQ IDNO. 10 or the amino acid sequence homologue thereof.
 6. A nucleic acidmolecule comprising a nucleotide sequence encoding the first polypeptidechain in the humanized anti-CD22 recombinant immunotoxin according toclaim 1 and/or a nucleotide sequence encoding the second polypeptidechain in the humanized anti-CD22 recombinant immunotoxin.
 7. A vectorcomprising the nucleic acid molecule according to claim
 6. 8. A hostcell transformed or transfected with the nucleic acid molecule accordingto claim 6 or a vector comprising the nucleic acid molecule.
 9. Acomposition comprising the humanized anti-CD22 recombinant immunotoxinaccording to claim
 1. 10. (canceled)
 11. A method for treating aCD22-related B-cell malignant tumor, comprising administering to asubject in need thereof the humanized anti-CD22 recombinant immunotoxinaccording to claim 1 or a composition comprising the humanized anti-CD22recombinant immunotoxin.
 12. A humanized anti-CD22 antibody or antibodyfragment, the antibody or antibody fragment comprising a heavy chainvariable region (V_(H)) and a light chain variable region (V_(L)),wherein the heavy chin variable region (V_(H)) comprises a amino acidsequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 or an amino acidsequence homologue thereof having at least 75% identity to the aminoacid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4, and the lightchain variable region (V_(L)) comprises an amino acid sequence as shownin SEQ ID NO. 16 or SEQ ID NO. 18 or an amino acid sequence homologuethereof having at least 75% identity to the amino acid sequence as shownin SEQ ID NO. 16 or SEQ ID NO.
 18. 13. A method for treating aCD22-related B-cell malignant tumor, comprising administering to asubject in need thereof one of: the nucleic acid molecule according toclaim 6; or a vector comprising the nucleic acid molecule; or a hostcell transformed or transfected with the nucleic acid molecule or thevector.
 14. The method for treating a CD22-related B-cell malignanttumor according to claim 11, wherein the CD22-related B-cell malignanttumor is a lymphoma or leukemia with high CD22 expression.
 15. Themethod for treating a CD22-related B-cell malignant tumor according toclaim 14, wherein the lymphoma is a non-Hodgkin's lymphoma, a smalllymphocytic lymphoma, or a mantle cell lymphoma; and the leukemia is achronic lymphocytic leukemia, a hairy cell leukemia, or an acutelymphocytic leukemia.
 16. The method for treating a CD22-related B-cellmalignant tumor according to claim 11, wherein the subject is a mammal.17. The method for treating a CD22-related B-cell malignant tumoraccording to claim 16, wherein the mammal is a human or non-humanprimate, a dog, a cat, a cow, a pig, a sheep, a horse, a murine, or arabbit.
 18. The method for treating a CD22-related B-cell malignanttumor according to claim 13, wherein the CD22-related B-cell malignanttumor is a lymphoma or leukemia with high CD22 expression.
 19. Themethod for treating a CD22-related B-cell malignant tumor according toclaim 18, wherein the lymphoma is a non-Hodgkin's lymphoma, a smalllymphocytic lymphoma, or a mantle cell lymphoma; and the leukemia is achronic lymphocytic leukemia, a hairy cell leukemia, or an acutelymphocytic leukemia.
 20. The method for treating a CD22-related B-cellmalignant tumor according to claim 13, wherein the subject is a mammal.21. The method for treating a CD22-related B-cell malignant tumoraccording to claim 20, wherein the mammal is a human or non-humanprimate, a dog, a cat, a cow, a pig, a sheep, a horse, a murine, or arabbit.